Mutant p53 and ELK-1 co-drive the expression of FRA-1 in breast cancer cells

Mutant p53 and ELK-1 co-drive the expression of FRA-1 in breast cancer cells | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Mutant p53 and ELK-1 co-drive the expression of FRA-1 in breast cancer cells Sike Hu, Manxue Wang, Ailing Ji, Jie Yang, Ruifang Gao, Xia Li, and 4 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-1452900/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Tumor-related p53 mutations can provoke activities different from p53 wild-type tumors to lose tumor-suppressing function. Cells harboring p53 mutations possess a more aggressive property associated with highly pro-invasion, pro-metastasis, proliferation, and cell survival. By comparing the gene expression profiles of p53 mutant and p53 knockdown cancer cells, we figure out FRA-1 as a potential effector of mutp53-mediated metastasis. We demonstrate that the expression of FRA-1, a gatekeeper of mesenchymal-epithelial transition encoded by FOSL1, elevates in the presence of p53 mutations. Mechanistically, mutant p53 binds to ELK-1 in BRCA cells, implying their collaboration to induce FRA-1 expression. In short, this study deciphers new insights into how mtp53 handles metastasis. Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Introduction TP53 in its wild-type form exerts a suppressive function in tumors. Wild-type p53 is involved in cellular senescence, cell cycle arrest, genome maintenance, apoptosis, and DNA repair in the active state. TP53 mutation is the leading cause of human cancer cells 1 . The general genetic alterations of p53 compromise the activity of wild-type p53, which is called loss of function (LOF) 2 . And it exerts a dominant-negative (DN) function against the remaining wild-type p53. In addition, there is increasing evidence that mounts p53 mutants exhibit gain-of-function (GOF) characteristics, by which mtp53 possesses "carcinogenic" properties and leads to a more aggressive tumor phenotype 3 . Mutant p53 (mtp53) is an effective metastasis regulator. Much evidence proves that mtp53 leads to the roughly similar acquisition of invasive and metastatic activities. To destruct epithelial polarity and reduce the integrity of cell-cell connections, mtp53 favors EMT programs by upregulating specific TFs such as ZEB1, ZEB2, SNAIL, SLUG 4 – 6 . Besides, several RTK pathways such as EGFR, AKT, PDGFRβ, and HGF/MET were induced by mutant p53 to promote tumor invasion and metastasis 7 . Mtp53 plays a vital role in the spatial regulation of RhoA activity by inducing MYO10. By harnessing the endocytic recycling machinery, mtp53 enhances the transport of integrin to the plasma membrane and promotes the invasion and movement of tumor cells 8 , 9 . Moreover, mtp53 upregulates ENTPD5 expression to promote N-glycosylated membrane protein folding, eventually fostering lung metastasis 10 . Further research demonstrated that mtp53 variants promote metastasis via miRNA 11 – 13 . Mutp53 protein has a new function of carcinogenesis and promoting cancer metastasis which depends on protein-protein interactions between mtp53 and other binding partners, including TFs such as Sp1 and ETS, and p53 family members such as p73 and p63 14–18 . FRA-1 belongs to the activator protein 1 (AP-1) family and is considered as a critical mediator of EMT/MET balance in tumor cells 19 – 22 . It involves cell motility programs through its ability to control several genes encoding EMT-TFs (e.g., ZEB1, TWIST), cell-cell adhesion proteins (CD44), and extracellular matrix-degrading enzymes (MMP1, MMP9) 23 – 27 . FRA-1 is a hub, resonding to various signals such as PKCθ /SPAK1, mTORC1/S6K1, and ERK 28 – 31 . In this study, we identify mtp53 as a driver of FRA-1. We analyzed the gene expression profile in metastatic BRAC cells to unravel the potential mechanism. We found a higher enrichment of FOSL1 (encoding FRA-1) in metastatic mutant p53 MDA-MB-231 cells than mutant p53 knockdown samples. In addition, we determined that FRA-1 expression depends on mtp53. In terms of mechanism, we discovered that mtp53 physically interacts with ELK-1 in MDA-MB-231 cells. Moreover, we disclosed that mtp53 and ELK-1 are recruited to the FOSL1 promoter together, suggesting they cooperate to induce FRA-1 expression. Additionally, in a comprehensive pan-cancer analysis, there was a highly significant correlation between FRA-1 expression and mutant p53. In conclusion, this study highlights that FRA-1 is a novel target for mtp53 to promote cell migration, providing new insights into how mtp53 navigates the tumor metastasis process and opens up a new way for transformation therapy. Materials And Methods shRNA and generation of stable cells. MDA-MB-231, T47D, and 293T cells were purchased from ATCC. We generated lentiviruses (biosettia) with a single shRNA targeting p53 or Elk-1 (Table S1) to knock down p53 and Elk-1 in MDA-MB-231 and T47D cells. The infected MDA-MB-231 and T47D cells were selected using a medium supplemented with 10 mg/mL puromycin (Sigma Aldrich). qRT-PCR and ChIP-qPCR. Total RNA was isolated with Trizol reagent (Invitrogen, USA). Then, the RNA (1µg) was synthesized into cDNA using a reverse transcription kit (Qiagen). The ChIP-DNA was prepared using an anti- ELK-1 antibody in MDA-MB-231 as described. All gene expression data were normalized with an internal control gene (GADPH). All ChIP-qPCR data was normalized with target amplification site in input. Primer sequences were listed in Supplementary Table S2& Table S3. Antibody. Antibodies were p53(#48818, Cell Signaling Technology), FRA-1(#5281, Cell Signaling Technology), ELK-1(#9182, Cell Signaling Technology), E-cadherin (#3195, Cell Signaling Technology), andβ-actin (#4970, Cell Signaling Technology). Immunofluorescent staining. Fix cells with 4% formaldehyde/PBS. After overnight incubation with anti-E-cadherin (1:200) at 4°C, the target protein was detected by anti-rabbit IgG PE-conjugated secondary antibody. Nuclei were stained with DAPI. Wound-healing assay. Cells in 6-well plates were grown to confluence. Use a 200 ml pipette tip to draw a line from the monolayer to remove part of the cells. The area of migrated cells was estimated after 24 hours and analyzed with ImageJ software. Transwell assay. For transwell migration assays, we seeded cells into the top chamber of a 24-well cell culture insert (Corning, 3422) with 1% FBS medium and then added 10% FBS medium to the bottom chamber. Cells transferred to the bottom of the membrane were fixed with 4% formaldehyde, stained with 0.05% crystal violet several hours later (MDA-MB-231, 17 hours; T47D, 45 hours), and counted. Measurements were performed in triplicates. GSEA enrichment analysis. Gene Set Enrichment Analysis (GSEA) using the expression matrix of differential genes in control and Tp53 knockdown RNA-seq data, the selected reference gene sets were c2.cp.reactome.v7.5.1.symbols.gmt and c2.cp. kegg.v7.5.symbols.gmt. The ggplot2 package is used to visualize GSEA collections. Analysis of TCGA gene expression data . To assess whether FRA-1 expression is induced in cancers with TP53 mutations, we used data from The Cancer Genome Atlas (TCGA). First, using XenaPython, we downloaded data from all samples in TCGA for BRCA, LUAD, and PAAD cancer types and classified the data, including wild-type p53 and p53GOF (missense mutations, including six hotspots ((R175, G245, R248, R249, R273, R282) together with R280K and L194F). GraphPad generated dot plots representing the distribution of gene expression values. Dot plots represent three genes in Tp53WT and Tp53GOF tumors from each cancer. The number of samples in each group was as follows: BRCA-p53WT = 675, Tp53GOF = 56; LUAD-p53WT = 276, Tp53GOF = 22; PAAD-p53WT = 57, Tp53GOF = 21. Results Mtp53 maintains EMT by enhancing the expression of a group of EMT-TFs MDA-MB-231 cells are a classic cell model of TNBC, and their transformation phenotype depends on the high-level R280K mutant variant of p53. To better comprehend the potential mechanism of mtp53 GOF, we used a well-designed and previously published MDA-MB-231 expression profile analysis data set 32 . Rather than examining one possible p53 gene at a time, GSEA analysis can identify sets of genes that represent molecular pathways and associated functions. GSEA analysis has demonstrated that the knockdown of p53 in the MDA-MB-231 cell line determines the EMT program. Among the gene groups, which are displaying the lowest NES value, are those associated with EMT. These include gene sets marked as KEGG pathway’s ECM receptor interaction (NES= -1.555113), TGF-β signaling pathway (NES=-1.4465687), basal cell carcinoma (NES=-1.4356328), Reactome’s degradation of the extracellular matrix (NES=-2.2472658), activation of matrix metalloproteinases (NES=-2.2133317), collagen degradation (NES=-2.133577), extracellular matrix organization (NES=-2.0230956), TGF-β receptor signaling in epithelial to mesenchymal transition (NES=-1.8143506), TGF-β receptor signaling activates SMADs (NES= -1.6533562) (Fig. 1 A,B). The bulk mainly refers to the same genes essential for EMT transformation. The GSEA analysis focused on EMT-related genes whose expression is reduced or induced upon EMT. Tissue inhibitor of metalloproteinase (TIMP-1, TIMP-2) is a natural inhibitor of matrix metalloproteinase (MMP). MMP increases after p53 gene knockout in MDA-MB-231 cells. In contrast, those mediate cell-to-cell and cell-to-matrix interactions molecular (THBS1, THBS2) reduced in p53 shRNA-expressing MDA-MB-231 cells. The reduction of mesenchymal genes, including VIM encoding vimentin, was also observed. Importantly, in response to mtp53 knockdown, a global decrease in a panel of EMT-TFs such as SNAIL, ZEB1, ZEB2, YAP1, JUN was detected (Fig. 1 C). Nevertheless, consistent with previous studies, our observations suggest that mtp53 is critical for maintaining EMT in BRAC. Mtp53 endows cells with mesenchymal traits and promote FRA-1 expression The GSEA analysis results show that mtp53 is involved in tumor invasion and metastasis. To examine this possibility, we investigated the mobility and transcriptional changes induced by mtp53 knockdown in MDA-MB-231 and T47D cells. To analyze the effect of mtp53 on cell migration, we performed wound-healing assays and transwell assays with MDA-MB-231 and T47D cells of mtp53 knockdown. From the results of these experiments, we can observe that mtp53 silencing decreases cell mobility (Fig. 2 A). Immunofluorescence staining of MDA-MB-231 and T47D cells showed that cell-cell adhesion involving E-cadherin was induced upon mtp53 knockdown (Fig. 2 B). These results indicated that mtp53 reduces the expression of E-cadherin and the formation of cell-cell adhesion, suggesting that mtp53 is essential for the maintenance of the mesenchymal characteristics of BRAC cells. Taken together, mtp53 is vital for cell mobility. According to the heat map, we also observed an increase in FOSL1. Given the importance of FRA-1 in EMT balance, we would like to know whether mtp53 maintains EMT through FRA-1. To test this possibility, we studied the transcriptional changes of FRA-1 caused by mtp53 gene knockdown in MDA-MB-231 and T47D cells. As we estimated, knockdown of mtp53 with two independent shRNAs preferentially caused loss of FRA-1 (Fig. 2 C, D). These results support the hypothesis that FRA-1 is vital to maintain the mesenchymal characteristics of cells harboring p53 mutations. Mtp53 is a direct activator of FOSL1 Some of the mtp53 regulatory genes may be directly regulated by mtp53, while others may be indirectly regulated. To determine whether FOSL1 is directly controlled by mtp53, we leverage p53 ChIP-seq data to check whether mtp53 binds to FOSL1. 699 p53 DTGs identified in MDA-MB-231 cells were compared with the p53 direct target genes (DTGs) identified in A549 and HEPG2 cells. The results showed that 463 genes, including FOSL1, were shared by three cell lines (Fig. 3 A). These data indicate that p53 target genes have little tissue specificity, and even artificial mutations occur in cells (HepG2 and A549). Using MDA-MB-231 p53 ChIP-seq data 32 , we found that p53 binds to four regions of the intron of the FOSL1 locus, all of which harbor response elements that firmly match the consensus binding sequence of p53. It is worth noting that p53 binding is conserved in the p53 ChIP-seq dataset generated from A549 and HepG2, although p53 has only one binding site in HepG2 cells. We examined the genome-wide distribution of mtp53. Three representative mtp53 binding peaks were selected in the crucial range(Fig. 3 B). ChIP-qPCR results confirm that mtp53 binds to the genomic region of FOSL1 in T47D cells and MDA-MB-231(Fig. 3 C). In conclusion, these data suggest that FOSL1 is a direct target of mtp53. ELK1- mtp53 cooperation is required for FRA-1 Expression The transcriptional function of mtp53 is related to its interaction with other transcription factors, thereby interrupting or enhancing its target genes. Under certain conditions, mtp53 can increase the activity of transcription factor partners, form transcription factor complexes with them, and be recruited to targeted promoters 18 . To elucidate the molecular mechanism by which p53 forms unique transcriptional complex factors in p53 mutant cells, we investigated the genome-wide distribution of other transcription factors using ChIP-seq data sets. Analyzing the synergy between p53 and other transcription factors will help to understand the characteristics of p53 synergy in specific cellular contexts. Enhancers strongly regulate FOSL1 transcription in its first intron, which contains some binding elements. Therefore, we manually searched UCSC for binding sites in the first intron of FOSL1 with these published ChIP-seq data. It was found that a large number of TFs, such as ELK-1, JUN, and JUNB, were recruited to this region. Therefore, we chose ELK-1 for further analysis, with the highest clustering score of 1000 (total score of 1000). ELK-1 was recruited into active chromatin labeled by H3K27ac and H3K4me3 deposition (Fig. 4 A) in Hela-s3 cells, which was close to p53 occupation, highlighting the functional interaction between ELK-1 and p53. The region was also found in MCF-7 breast cancer cells, K562 leukemia cells, and GM12878 cells (not shown). Furthermore, co-immunoprecipitation (Co-IP) with p53 antibody from MDA-MB-231 cells and T47D cells revealed the interaction between mtp53 and ELK-1 in both cell lines (Fig. 4 B). Knockdown experiments in MDA-MB-231 cells showed that ELK-1 made a significant contribution to FOSL1 expression (Fig. 4 C, D). A considerable contribution of ELK-1 to FOSL1 expression was also inspected in T47D cells (Fig. 4 C, D), suggesting that p53-ELK1 cooperation is usually crucial in p53 mutant cells. FRA-1 Expression Levels Correlate with GOF mtp53 in Human Tumor Samples Tumor Genome Atlas (TCGA) RNA-sequencing data provided us with a novel approach to probe the correlation between TP53 GOF missense mutations and FRA-1 expression. TP53 GOF missense mutations are prevalent in multiple cancer types. To test the effect of TP53 GOF on the downstream expression of FRA-1 (compared with SNAIL and SLUG), we observed the expression of these genes in three types of cancer prevalent in the TP53 mutation: breast cancer (BRCA), lung adenocarcinoma (LUAD) and pancreatic cancer (PAAD). We divided the cases into two classes for each cancer according to their p53 mutation status as wild-type p53 and p53GOF (missense mutation including R175, G245, R248, R249 R273, R282, R280K, and L194F). As Zhu pointed out in the study, other p53 mutations (other missense mutations, frame insertion/deletions, or splice mutations) and null p53 (p53 nonsense mutations or frameshift truncations) were not included in the further analysis because of significant differences in p53 function 33 . The impact is unpredictable. Then, we compared the expression of SNAIL, SLUG , and FRA-1 between the two classes in the three cancer types. Consistent with mtp53-dependent expression, FRA-1 RNA levels were significantly higher in cases with p53GOF mutations than wild-type p53 cases (Fig. 5 ). Similar but less robust patterns were observed for SNAIL and SLUG expression in both cases (Fig. 5 ). Thus, p53 GOF mutations are associated with high levels of FRA-1 expression in a wide range of patient groups with different tumor entities. Discussion The prevalent TP53 mutations in human cancer have promoted the development of targeted therapy for the TP53 pathway 34 . The realization of TP53-based treatment depends on a comprehensive understanding of the mechanism of tumor-related TP53 mutation. Through gene expression profiling of control & knockdown for Tp53 of MDA-MB-231 cells, we identified FOSL1 as a novel mediator of mtp53. We demonstrated for the first time that FOSL1 expression directly depended on mtp53, indicating that FRA-1 may be a novel mutant p53 effector. The comparative results of shRNA experiments in MDA-MB-231 and T47D cells showed that FRA-1 might be one of the crucial targets of mtp53 in BRAC. We demonstrated that mtp53 interacted directly with ELK-1 and their synergistic effect induces FOSL1 gene expression. High levels of FRA-1 expression are associated with p53GOF mutations in multiple tumors, which may underlie a possible basis for common cancer metastasis. This study regarded FRA-1 as a novel downstream effector of mutated p53 that favored tumor metastasis. FRA-1 establishes and maintains the EMT program in different cancer types by directly regulating the expression of EMT-TFs 22 . In breast epithelial cells, RAS / ERK2 increases the expression of ZEB1 and ZEB2 by driving FRA-1 upregulation, resulting in complete EMT 35 . In malignant melanoma cells, it is shown that FRA-1 binds directly to AP-1 binding elements located in TWIST1, SNAIL2, ZEB1 , and ZEB2 gene promoters 27 . The Weinberg lab's findings reinforce the central role of FRA-1 in EMT. FOSL1 is directly motivated by TWIST1 and SNAIL1, thereby acting as an effector of the EMT pathway 36 . Furthermore, FOSL1 serves as a hub that brings together numerous upstream regulatory pathways, including oncogenes of the MEK-ERK module and tumor suppressors such as p53, APC, and PTEN 22 . Here, we illustrated that mtp53 interacted with ELK-1 and regulated FRA-1 expression by binding to the FOSL1 promoter, ultimately promoting tumor metastasis. The control role of ELK-1 on the migration of several human breast cells has been demonstrated, suggesting that ELK-1 plays a part in the metastasis of breast cancer cells 37 . Genome-wide analysis showed that most of the genes targeted by ELK-1 were related to cell migration and actin cytoskeleton 38 . In addition, ELK-1 was recruited to the ETS binding site of the MMP-9 promoter, thus enhancing its transcription 39 . There are many TFs occupied on the promoter of FOSL1, including ELK-1, SRF, AP-1, and ATF/CREB 35 . Although we cannot formally exclude additional partners of mtp53, ELK-1 may be a significant recruitment factor because the consumption of ELK-1 impairs FRA-1 expression in BRAC. In light of the role of FRA-1 in metastasis, targeting FRA-1 in aggressive BRCA may be a new therapeutic option. This study offers a prism through which to look at the molecular basis of mtp53-driven metastasis and an identification scheme for a novel p53 mutational metastatic therapeutic target. Declarations Data availability The datasets analyzed during the current study are available as follows. Gene expression microarray data can be accessed under GEO Accession No. GSE68248. ChIP-seq data were obtained from GEO Accession No. GSE66543 (MDA-MB-231) and ENCODE Project Consortium: A549 (ENCSR112XUO), HepG2 (ENCSR980EGJ), and Hela-s3 (ENCSR454DOC, ENCSR717QSS, ENCSR068MRQ). Author contributions S.H. and H.L. designed the experiments, performed the statistical analysis, and wrote the manuscript. M.W., A. J., J.Y., R. G., X.L., and L.S performed experiments acquired. Z.Y. and Y.Z. assisted with writing the manuscript. All authors reviewed and approved the final version of the manuscript. Competing interests The authors declare no competing interests. References Kandoth, C. et al. Mutational landscape and significance across 12 major cancer types. Nature 502 , 333-339, doi:10.1038/nature12634 (2013). Rivlin, N., Brosh, R., Oren, M. & Rotter, V. 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Bradykinin induces matrix metalloproteinase-9 expression and cell migration through a PKC-delta-dependent ERK/Elk-1 pathway in astrocytes. Glia 56 , 619-632, doi:10.1002/glia.20637 (2008). Additional Declarations No competing interests reported. Supplementary Files supplementary.docx Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-1452900","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":91197267,"identity":"1bab7bf3-a99b-44a6-b6e7-b0a75ba1fad6","order_by":0,"name":"Sike Hu","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAArUlEQVRIiWNgGAWjYBACAyA+2MBgw8PP3kCaljQZyZ4DJGhhbGA4bGNww4FILeYS6Q8Pzqg4z8Nwg4Hxw8ccIrRYzsgxOLjhzG0extkNzJIztxHjsBs5DAcftt3mYZY5wMbMS5yW9AcHH/47x8MmkUC0lgSDgxsbDvDwEK/lzBuDgzOOJfNI8BxsJtIvx9Mff+ypsbO3P9588MNHYrQgAWD0jIJRMApGwSigEgAAtOU7lwd3S9EAAAAASUVORK5CYII=","orcid":"","institution":"Tianjin Institute of Medical \u0026 Pharmaceutical Sciences","correspondingAuthor":true,"submittingAuthor":false,"prefix":"","firstName":"Sike","middleName":"","lastName":"Hu","suffix":""},{"id":91197268,"identity":"98e24d18-7e8b-4dd4-b980-a58ccb5e785a","order_by":1,"name":"Manxue Wang","email":"","orcid":"","institution":"Tianjin Institute of Medical \u0026 Pharmaceutical Sciences","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Manxue","middleName":"","lastName":"Wang","suffix":""},{"id":91197269,"identity":"93113356-3ec8-4ea8-88cb-74c9b75b50ef","order_by":2,"name":"Ailing Ji","email":"","orcid":"","institution":"Tianjin Medicine and Health Research Center","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Ailing","middleName":"","lastName":"Ji","suffix":""},{"id":91197270,"identity":"ddb05a95-55c4-44c2-ac2b-b9f137f29904","order_by":3,"name":"Jie Yang","email":"","orcid":"","institution":"Tianjin Medicine and Health Research Center","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Jie","middleName":"","lastName":"Yang","suffix":""},{"id":91197271,"identity":"070079de-ae7d-41f9-8396-8550f3909de4","order_by":4,"name":"Ruifang Gao","email":"","orcid":"","institution":"Tianjin Institute of Medical \u0026 Pharmaceutical Sciences","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Ruifang","middleName":"","lastName":"Gao","suffix":""},{"id":91197272,"identity":"60590b20-2b9d-40a2-aa1b-89d361aaed8c","order_by":5,"name":"Xia Li","email":"","orcid":"","institution":"Tianjin Institute of Medical \u0026 Pharmaceutical Sciences","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Xia","middleName":"","lastName":"Li","suffix":""},{"id":91197273,"identity":"1cded9e8-d64e-4761-81ea-d63ee9c5b6a6","order_by":6,"name":"Lili Sun","email":"","orcid":"","institution":"Tianjin Medicine and Health Research Center","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Lili","middleName":"","lastName":"Sun","suffix":""},{"id":91197274,"identity":"fd2cb5d2-7b64-45b5-9bcb-09cbcea5af24","order_by":7,"name":"Zibo Yang","email":"","orcid":"","institution":"Tianjin Institute of Medical \u0026 Pharmaceutical Sciences","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Zibo","middleName":"","lastName":"Yang","suffix":""},{"id":91197275,"identity":"36cbe131-29f2-4210-9176-bd062abb158b","order_by":8,"name":"Ying Zhang","email":"","orcid":"","institution":"Tianjin Medicine and Health Research Center","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Ying","middleName":"","lastName":"Zhang","suffix":""},{"id":91197276,"identity":"8417dc33-f122-4948-9764-b9e28830b1da","order_by":9,"name":"Hongbin Liu","email":"","orcid":"","institution":"Tianjin Medicine and Health Research Center","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Hongbin","middleName":"","lastName":"Liu","suffix":""}],"badges":[],"createdAt":"2022-03-15 07:29:24","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-1452900/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-1452900/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":19343806,"identity":"549d0df1-d4c5-4c43-9306-b66696cdb87c","added_by":"auto","created_at":"2022-03-17 18:35:00","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":243063,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003ePathway and gene identified through GSEA. \u003c/strong\u003eBubble chart of KEGG enrichment (A) and Reactome enrichment (B) analysis on gse68249 dataset using GSEA. The bubble color changes from blue to red, indicating a gradual increase in significance. Heatmap shows the RNA sequencing results of arbitrarily selected EMT-related genes in MDA-MB-231(C), ignoring P value and fold change. Counts were normalized by Deseq2 (GSE68249).\u003c/p\u003e","description":"","filename":"floatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-1452900/v1/45f2782c70ce99a6b7f58e3b.png"},{"id":19343811,"identity":"a9342472-0a05-45e0-9240-b07f8c5d70ad","added_by":"auto","created_at":"2022-03-17 18:35:00","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":1631515,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eMtp53 promotes cell migration. \u003c/strong\u003eWound healing and transwell assay of mtp53-silenced MDA-MB-231 and T47D cells. Scrambled shRNA was used as control (A). All data were expressed as mean ± SD, p-value from student t-test, **p≤0.01; *p≤0.05。Immunofluorescence staining of E-cadherin in mtp53-silenced MDA-MB-231 and T47D cells (B). Scale bar, 50um. QRT-PCR analyses of the indicated transcripts (relative to GADPH)(C). mRNA expression is normalized to the expression of control cells. All data are presented as mean ± SD, **p≤0.01; *p≤0.05. Immunoblot analysis of FRA-1 in mtp53-silenced MDA-MB-231 and T47D cells (D).\u003c/p\u003e","description":"","filename":"floatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-1452900/v1/0638f016921337d3a8c44e1e.png"},{"id":19343809,"identity":"109faf8c-09e9-44f4-a11e-7f5aaef5d448","added_by":"auto","created_at":"2022-03-17 18:35:00","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":168058,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eTP53 directly controls FOSL1.\u003c/strong\u003e Comparison of mtp53 DTGs in three cancer cell lines from different tissues. Wayne diagram shows the number of mtp53 DTGs identified in HepG2, A549, and MDA-MB-231 cells (A). The numbers in the pie chart overlap represent shared genes. FOSL1 is shared among three cancer cell lines.\u003c/p\u003e\u003cp\u003e(B)ChIP-seq binding profile of mtp53 in A549. (C) ChIP-qPCR analysis of TP53 in MDA-MB-231 and T47D cells. Relative fold changes were normalized to normal rabbit serum (NRS). All data were expressed as mean ± SD. **p≤0.01; *p≤0.05\u003c/p\u003e","description":"","filename":"floatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-1452900/v1/4f9c92173d743a4a671deedd.png"},{"id":19344313,"identity":"75f4ae88-f688-45f5-a47b-e335bebfa6f1","added_by":"auto","created_at":"2022-03-17 18:38:00","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":300887,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eELK-1 acts as a cofactor of mtp53.\u003c/strong\u003e ChIP-seq profile of the FOSL1 locus. P53 chromatin binding in A549 cells, ELK-1 chromatin binding, H3K27ac\u0026amp; H3K4me3 deposition patterns in Hela-s3 cells were obtained from the ENCODE database (A). The protein extracts of MDA-MB-231 and T47D cells were immunoprecipitated (IP) with IgG or p53 antibody, and then p53 and ELK-1were immunoblotted (B). Q-PCR analyzed the indicated transcripts (relative to GADPH) on ELK-1 knockdown in MDA-MB-231 and T47D cells(C). All data are expressed as mean ± SD. **p≤0.01; *p≤0.05. Elk-1 and FRA-1 were detected by Western blot analysis on ELK-1 knockdown in MDA-MB-231 and T47D cells (D).\u003c/p\u003e","description":"","filename":"floatimage4.png","url":"https://assets-eu.researchsquare.com/files/rs-1452900/v1/18d9e79fa9b34b7a21240cfa.png"},{"id":19343808,"identity":"29fcb08a-3c25-4421-b81d-4b24e8e577fd","added_by":"auto","created_at":"2022-03-17 18:35:00","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":244136,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eFRA-1 induction in tumors with P53 mutations.\u003c/strong\u003e The gene expression values of SNAIL, SLUG, and FRA-1 in breast cancer (BRCA), lung adenocarcinoma (LUAD), and pancreatic cancer (PAAD). Expression values were based on Pan-cancer standardized RNA-seq data from The Cancer Genome Atlas (TCGA). Each point represents the expression level of a single tumor, and the horizontal line stands for the average expression of cancer/genotype population. Significant changes in Tp53GOF relative to Tp53WT are shown(Wilcoxon rank-sum test; **p≤0.01; *p≤0.05).\u003c/p\u003e","description":"","filename":"floatimage5.png","url":"https://assets-eu.researchsquare.com/files/rs-1452900/v1/b2122403ba7973143fccc292.png"},{"id":21212640,"identity":"08cc746c-15ee-4129-b25e-bb2b1c9153d6","added_by":"auto","created_at":"2022-05-09 05:59:22","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2501348,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-1452900/v1/4954ccc5-146d-43be-87ba-288c0f3ea96a.pdf"},{"id":19344312,"identity":"692148c2-5c2c-4498-946d-341b2174b7b0","added_by":"auto","created_at":"2022-03-17 18:38:00","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":17461,"visible":true,"origin":"","legend":"","description":"","filename":"supplementary.docx","url":"https://assets-eu.researchsquare.com/files/rs-1452900/v1/292db23f5b734fca78845780.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Mutant p53 and ELK-1 co-drive the expression of FRA-1 in breast cancer cells","fulltext":[{"header":"Introduction","content":"\u003cp\u003eTP53 in its wild-type form exerts a suppressive function in tumors. Wild-type p53 is involved in cellular senescence, cell cycle arrest, genome maintenance, apoptosis, and DNA repair in the active state. TP53 mutation is the leading cause of human cancer cells\u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e. The general genetic alterations of p53 compromise the activity of wild-type p53, which is called loss of function (LOF)\u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e. And it exerts a dominant-negative (DN) function against the remaining wild-type p53. In addition, there is increasing evidence that mounts p53 mutants exhibit gain-of-function (GOF) characteristics, by which mtp53 possesses \"carcinogenic\" properties and leads to a more aggressive tumor phenotype\u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eMutant p53 (mtp53) is an effective metastasis regulator. Much evidence proves that mtp53 leads to the roughly similar acquisition of invasive and metastatic activities. To destruct epithelial polarity and reduce the integrity of cell-cell connections, mtp53 favors EMT programs by upregulating specific TFs such as ZEB1, ZEB2, SNAIL, SLUG\u003csup\u003e\u003cspan additionalcitationids=\"CR5\" citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u003c/sup\u003e. Besides, several RTK pathways such as EGFR, AKT, PDGFRβ, and HGF/MET were induced by mutant p53 to promote tumor invasion and metastasis\u003csup\u003e\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u003c/sup\u003e. Mtp53 plays a vital role in the spatial regulation of RhoA activity by inducing MYO10. By harnessing the endocytic recycling machinery, mtp53 enhances the transport of integrin to the plasma membrane and promotes the invasion and movement of tumor cells\u003csup\u003e\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e,\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u003c/sup\u003e. Moreover, mtp53 upregulates ENTPD5 expression to promote N-glycosylated membrane protein folding, eventually fostering lung metastasis\u003csup\u003e\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e. Further research demonstrated that mtp53 variants promote metastasis via miRNA\u003csup\u003e\u003cspan additionalcitationids=\"CR12\" citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003e. Mutp53 protein has a new function of carcinogenesis and promoting cancer metastasis which depends on protein-protein interactions between mtp53 and other binding partners, including TFs such as Sp1 and ETS, and p53 family members such as p73 and p63\u003csup\u003e14\u0026ndash;18\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eFRA-1 belongs to the activator protein 1 (AP-1) family and is considered as a critical mediator of EMT/MET balance in tumor cells\u003csup\u003e\u003cspan additionalcitationids=\"CR20 CR21\" citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u003c/sup\u003e. It involves cell motility programs through its ability to control several genes encoding EMT-TFs (e.g., ZEB1, TWIST), cell-cell adhesion proteins (CD44), and extracellular matrix-degrading enzymes (MMP1, MMP9)\u003csup\u003e\u003cspan additionalcitationids=\"CR24 CR25 CR26\" citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u003c/sup\u003e. FRA-1 is a hub, resonding to various signals such as PKCθ /SPAK1, mTORC1/S6K1, and ERK\u003csup\u003e\u003cspan additionalcitationids=\"CR29 CR30\" citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e\u003c/sup\u003e. In this study, we identify mtp53 as a driver of FRA-1. We analyzed the gene expression profile in metastatic BRAC cells to unravel the potential mechanism. We found a higher enrichment of FOSL1 (encoding FRA-1) in metastatic mutant p53 MDA-MB-231 cells than mutant p53 knockdown samples. In addition, we determined that FRA-1 expression depends on mtp53. In terms of mechanism, we discovered that mtp53 physically interacts with ELK-1 in MDA-MB-231 cells. Moreover, we disclosed that mtp53 and ELK-1 are recruited to the FOSL1 promoter together, suggesting they cooperate to induce FRA-1 expression. Additionally, in a comprehensive pan-cancer analysis, there was a highly significant correlation between FRA-1 expression and mutant p53.\u003c/p\u003e \u003cp\u003eIn conclusion, this study highlights that FRA-1 is a novel target for mtp53 to promote cell migration, providing new insights into how mtp53 navigates the tumor metastasis process and opens up a new way for transformation therapy.\u003c/p\u003e"},{"header":"Materials And Methods","content":"\u003cp\u003e \u003cb\u003eshRNA and generation of stable cells.\u003c/b\u003e MDA-MB-231, T47D, and 293T cells were purchased from ATCC. We generated lentiviruses (biosettia) with a single shRNA targeting p53 or Elk-1 (Table S1) to knock down p53 and Elk-1 in MDA-MB-231 and T47D cells. The infected MDA-MB-231 and T47D cells were selected using a medium supplemented with 10 mg/mL puromycin (Sigma Aldrich).\u003c/p\u003e \u003cp\u003e \u003cb\u003eqRT-PCR and ChIP-qPCR.\u003c/b\u003e Total RNA was isolated with Trizol reagent (Invitrogen, USA). Then, the RNA (1\u0026micro;g) was synthesized into cDNA using a reverse transcription kit (Qiagen). The ChIP-DNA was prepared using an anti- ELK-1 antibody in MDA-MB-231 as described. All gene expression data were normalized with an internal control gene (GADPH). All ChIP-qPCR data was normalized with target amplification site in input. Primer sequences were listed in Supplementary Table S2\u0026amp; Table S3.\u003c/p\u003e \u003cp\u003e \u003cb\u003eAntibody.\u003c/b\u003e Antibodies were p53(#48818, Cell Signaling Technology), FRA-1(#5281, Cell Signaling Technology), ELK-1(#9182, Cell Signaling Technology), E-cadherin (#3195, Cell Signaling Technology), andβ-actin (#4970, Cell Signaling Technology).\u003c/p\u003e \u003cp\u003e \u003cb\u003eImmunofluorescent staining.\u003c/b\u003e Fix cells with 4% formaldehyde/PBS. After overnight incubation with anti-E-cadherin (1:200) at 4\u0026deg;C, the target protein was detected by anti-rabbit IgG PE-conjugated secondary antibody. Nuclei were stained with DAPI.\u003c/p\u003e \u003cp\u003e \u003cb\u003eWound-healing assay.\u003c/b\u003e Cells in 6-well plates were grown to confluence. Use a 200 ml pipette tip to draw a line from the monolayer to remove part of the cells. The area of migrated cells was estimated after 24 hours and analyzed with ImageJ software.\u003c/p\u003e \u003cp\u003e \u003cb\u003eTranswell assay.\u003c/b\u003e For transwell migration assays, we seeded cells into the top chamber of a 24-well cell culture insert (Corning, 3422) with 1% FBS medium and then added 10% FBS medium to the bottom chamber. Cells transferred to the bottom of the membrane were fixed with 4% formaldehyde, stained with 0.05% crystal violet several hours later (MDA-MB-231, 17 hours; T47D, 45 hours), and counted. Measurements were performed in triplicates.\u003c/p\u003e \u003cp\u003e \u003cb\u003eGSEA enrichment analysis.\u003c/b\u003e Gene Set Enrichment Analysis (GSEA) using the expression matrix of differential genes in control and Tp53 knockdown RNA-seq data, the selected reference gene sets were c2.cp.reactome.v7.5.1.symbols.gmt and c2.cp. kegg.v7.5.symbols.gmt. The ggplot2 package is used to visualize GSEA collections.\u003c/p\u003e \u003cp\u003e \u003cb\u003eAnalysis of TCGA gene expression data\u003c/b\u003e. To assess whether FRA-1 expression is induced in cancers with TP53 mutations, we used data from The Cancer Genome Atlas (TCGA). First, using XenaPython, we downloaded data from all samples in TCGA for BRCA, LUAD, and PAAD cancer types and classified the data, including wild-type p53 and p53GOF (missense mutations, including six hotspots ((R175, G245, R248, R249, R273, R282) together with R280K and L194F). GraphPad generated dot plots representing the distribution of gene expression values. Dot plots represent three genes in Tp53WT and Tp53GOF tumors from each cancer. The number of samples in each group was as follows: BRCA-p53WT\u0026thinsp;=\u0026thinsp;675, Tp53GOF\u0026thinsp;=\u0026thinsp;56; LUAD-p53WT\u0026thinsp;=\u0026thinsp;276, Tp53GOF\u0026thinsp;=\u0026thinsp;22; PAAD-p53WT\u0026thinsp;=\u0026thinsp;57, Tp53GOF\u0026thinsp;=\u0026thinsp;21.\u003c/p\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003eMtp53 maintains EMT by enhancing the expression of a group of EMT-TFs\u003c/h2\u003e \u003cp\u003eMDA-MB-231 cells are a classic cell model of TNBC, and their transformation phenotype depends on the high-level R280K mutant variant of p53. To better comprehend the potential mechanism of mtp53 GOF, we used a well-designed and previously published MDA-MB-231 expression profile analysis data set \u003csup\u003e\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e\u003c/sup\u003e. Rather than examining one possible p53 gene at a time, GSEA analysis can identify sets of genes that represent molecular pathways and associated functions. GSEA analysis has demonstrated that the knockdown of p53 in the MDA-MB-231 cell line determines the EMT program. Among the gene groups, which are displaying the lowest NES value, are those associated with EMT. These include gene sets marked as KEGG pathway\u0026rsquo;s ECM receptor interaction (NES= -1.555113), TGF-β signaling pathway (NES=-1.4465687), basal cell carcinoma (NES=-1.4356328), Reactome\u0026rsquo;s degradation of the extracellular matrix (NES=-2.2472658), activation of matrix metalloproteinases (NES=-2.2133317), collagen degradation (NES=-2.133577), extracellular matrix organization (NES=-2.0230956), TGF-β receptor signaling in epithelial to mesenchymal transition (NES=-1.8143506), TGF-β receptor signaling activates SMADs (NES= -1.6533562) (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA,B). The bulk mainly refers to the same genes essential for EMT transformation.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThe GSEA analysis focused on EMT-related genes whose expression is reduced or induced upon EMT. Tissue inhibitor of metalloproteinase (TIMP-1, TIMP-2) is a natural inhibitor of matrix metalloproteinase (MMP). MMP increases after p53 gene knockout in MDA-MB-231 cells. In contrast, those mediate cell-to-cell and cell-to-matrix interactions molecular (THBS1, THBS2) reduced in p53 shRNA-expressing MDA-MB-231 cells. The reduction of mesenchymal genes, including VIM encoding vimentin, was also observed. Importantly, in response to mtp53 knockdown, a global decrease in a panel of EMT-TFs such as SNAIL, ZEB1, ZEB2, YAP1, JUN was detected (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC). Nevertheless, consistent with previous studies, our observations suggest that mtp53 is critical for maintaining EMT in BRAC.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003eMtp53 endows cells with mesenchymal traits and promote FRA-1 expression\u003c/h2\u003e \u003cp\u003eThe GSEA analysis results show that mtp53 is involved in tumor invasion and metastasis. To examine this possibility, we investigated the mobility and transcriptional changes induced by mtp53 knockdown in MDA-MB-231 and T47D cells. To analyze the effect of mtp53 on cell migration, we performed wound-healing assays and transwell assays with MDA-MB-231 and T47D cells of mtp53 knockdown. From the results of these experiments, we can observe that mtp53 silencing decreases cell mobility (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA). Immunofluorescence staining of MDA-MB-231 and T47D cells showed that cell-cell adhesion involving E-cadherin was induced upon mtp53 knockdown (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB). These results indicated that mtp53 reduces the expression of E-cadherin and the formation of cell-cell adhesion, suggesting that mtp53 is essential for the maintenance of the mesenchymal characteristics of BRAC cells. Taken together, mtp53 is vital for cell mobility.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eAccording to the heat map, we also observed an increase in FOSL1. Given the importance of FRA-1 in EMT balance, we would like to know whether mtp53 maintains EMT through FRA-1. To test this possibility, we studied the transcriptional changes of FRA-1 caused by mtp53 gene knockdown in MDA-MB-231 and T47D cells. As we estimated, knockdown of mtp53 with two independent shRNAs preferentially caused loss of FRA-1 (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC, D). These results support the hypothesis that FRA-1 is vital to maintain the mesenchymal characteristics of cells harboring p53 mutations.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003eMtp53 is a direct activator of FOSL1\u003c/h2\u003e \u003cp\u003eSome of the mtp53 regulatory genes may be directly regulated by mtp53, while others may be indirectly regulated. To determine whether FOSL1 is directly controlled by mtp53, we leverage p53 ChIP-seq data to check whether mtp53 binds to FOSL1. 699 p53 DTGs identified in MDA-MB-231 cells were compared with the p53 direct target genes (DTGs) identified in A549 and HEPG2 cells. The results showed that 463 genes, including FOSL1, were shared by three cell lines (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA). These data indicate that p53 target genes have little tissue specificity, and even artificial mutations occur in cells (HepG2 and A549).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eUsing MDA-MB-231 p53 ChIP-seq data\u003csup\u003e\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e\u003c/sup\u003e, we found that p53 binds to four regions of the intron of the FOSL1 locus, all of which harbor response elements that firmly match the consensus binding sequence of p53. It is worth noting that p53 binding is conserved in the p53 ChIP-seq dataset generated from A549 and HepG2, although p53 has only one binding site in HepG2 cells. We examined the genome-wide distribution of mtp53. Three representative mtp53 binding peaks were selected in the crucial range(Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB). ChIP-qPCR results confirm that mtp53 binds to the genomic region of FOSL1 in T47D cells and MDA-MB-231(Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC). In conclusion, these data suggest that FOSL1 is a direct target of mtp53.\u003c/p\u003e \u003cdiv id=\"Sec7\" class=\"Section3\"\u003e \u003ch2\u003eELK1- mtp53 cooperation is required for FRA-1 Expression\u003c/h2\u003e \u003cp\u003eThe transcriptional function of mtp53 is related to its interaction with other transcription factors, thereby interrupting or enhancing its target genes. Under certain conditions, mtp53 can increase the activity of transcription factor partners, form transcription factor complexes with them, and be recruited to targeted promoters\u003csup\u003e\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003e. To elucidate the molecular mechanism by which p53 forms unique transcriptional complex factors in p53 mutant cells, we investigated the genome-wide distribution of other transcription factors using ChIP-seq data sets. Analyzing the synergy between p53 and other transcription factors will help to understand the characteristics of p53 synergy in specific cellular contexts.\u003c/p\u003e \u003cp\u003eEnhancers strongly regulate FOSL1 transcription in its first intron, which contains some binding elements. Therefore, we manually searched UCSC for binding sites in the first intron of FOSL1 with these published ChIP-seq data. It was found that a large number of TFs, such as ELK-1, JUN, and JUNB, were recruited to this region. Therefore, we chose ELK-1 for further analysis, with the highest clustering score of 1000 (total score of 1000). ELK-1 was recruited into active chromatin labeled by H3K27ac and H3K4me3 deposition (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA) in Hela-s3 cells, which was close to p53 occupation, highlighting the functional interaction between ELK-1 and p53. The region was also found in MCF-7 breast cancer cells, K562 leukemia cells, and GM12878 cells (not shown). Furthermore, co-immunoprecipitation (Co-IP) with p53 antibody from MDA-MB-231 cells and T47D cells revealed the interaction between mtp53 and ELK-1 in both cell lines (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eKnockdown experiments in MDA-MB-231 cells showed that ELK-1 made a significant contribution to FOSL1 expression (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC, D). A considerable contribution of ELK-1 to FOSL1 expression was also inspected in T47D cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC, D), suggesting that p53-ELK1 cooperation is usually crucial in p53 mutant cells.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eFRA-1 Expression Levels Correlate with GOF mtp53 in Human Tumor Samples\u003c/h2\u003e \u003cp\u003eTumor Genome Atlas (TCGA) RNA-sequencing data provided us with a novel approach to probe the correlation between TP53 GOF missense mutations and FRA-1 expression. TP53 GOF missense mutations are prevalent in multiple cancer types. To test the effect of TP53 GOF on the downstream expression of FRA-1 (compared with SNAIL and SLUG), we observed the expression of these genes in three types of cancer prevalent in the TP53 mutation: breast cancer (BRCA), lung adenocarcinoma (LUAD) and pancreatic cancer (PAAD).\u003c/p\u003e \u003cp\u003eWe divided the cases into two classes for each cancer according to their p53 mutation status as wild-type p53 and p53GOF (missense mutation including R175, G245, R248, R249 R273, R282, R280K, and L194F). As Zhu pointed out in the study, other p53 mutations (other missense mutations, frame insertion/deletions, or splice mutations) and null p53 (p53 nonsense mutations or frameshift truncations) were not included in the further analysis because of significant differences in p53 function \u003csup\u003e\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e\u003c/sup\u003e. The impact is unpredictable. Then, we compared the expression of \u003cem\u003eSNAIL, SLUG\u003c/em\u003e, and \u003cem\u003eFRA-1\u003c/em\u003e between the two classes in the three cancer types. Consistent with mtp53-dependent expression, \u003cem\u003eFRA-1\u003c/em\u003e RNA levels were significantly higher in cases with p53GOF mutations than wild-type p53 cases (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e). Similar but less robust patterns were observed for \u003cem\u003eSNAIL\u003c/em\u003e and \u003cem\u003eSLUG\u003c/em\u003e expression in both cases (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e). Thus, p53 GOF mutations are associated with high levels of \u003cem\u003eFRA-1\u003c/em\u003e expression in a wide range of patient groups with different tumor entities.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eThe prevalent TP53 mutations in human cancer have promoted the development of targeted therapy for the TP53 pathway\u003csup\u003e\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e\u003c/sup\u003e. The realization of TP53-based treatment depends on a comprehensive understanding of the mechanism of tumor-related TP53 mutation. Through gene expression profiling of control \u0026amp; knockdown for Tp53 of MDA-MB-231 cells, we identified FOSL1 as a novel mediator of mtp53. We demonstrated for the first time that FOSL1 expression directly depended on mtp53, indicating that FRA-1 may be a novel mutant p53 effector. The comparative results of shRNA experiments in MDA-MB-231 and T47D cells showed that FRA-1 might be one of the crucial targets of mtp53 in BRAC. We demonstrated that mtp53 interacted directly with ELK-1 and their synergistic effect induces FOSL1 gene expression. High levels of FRA-1 expression are associated with p53GOF mutations in multiple tumors, which may underlie a possible basis for common cancer metastasis.\u003c/p\u003e \u003cp\u003eThis study regarded FRA-1 as a novel downstream effector of mutated p53 that favored tumor metastasis. FRA-1 establishes and maintains the EMT program in different cancer types by directly regulating the expression of EMT-TFs\u003csup\u003e\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u003c/sup\u003e. In breast epithelial cells, RAS / ERK2 increases the expression of ZEB1 and ZEB2 by driving FRA-1 upregulation, resulting in complete EMT\u003csup\u003e\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e\u003c/sup\u003e. In malignant melanoma cells, it is shown that FRA-1 binds directly to AP-1 binding elements located in \u003cem\u003eTWIST1, SNAIL2, ZEB1\u003c/em\u003e, and \u003cem\u003eZEB2\u003c/em\u003e gene promoters \u003csup\u003e\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u003c/sup\u003e. The Weinberg lab's findings reinforce the central role of FRA-1 in EMT. FOSL1 is directly motivated by TWIST1 and SNAIL1, thereby acting as an effector of the EMT pathway\u003csup\u003e\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e\u003c/sup\u003e. Furthermore, FOSL1 serves as a hub that brings together numerous upstream regulatory pathways, including oncogenes of the MEK-ERK module and tumor suppressors such as p53, APC, and PTEN \u003csup\u003e\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eHere, we illustrated that mtp53 interacted with ELK-1 and regulated FRA-1 expression by binding to the FOSL1 promoter, ultimately promoting tumor metastasis. The control role of ELK-1 on the migration of several human breast cells has been demonstrated, suggesting that ELK-1 plays a part in the metastasis of breast cancer cells \u003csup\u003e\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e\u003c/sup\u003e. Genome-wide analysis showed that most of the genes targeted by ELK-1 were related to cell migration and actin cytoskeleton\u003csup\u003e\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e\u003c/sup\u003e. In addition, ELK-1 was recruited to the ETS binding site of the MMP-9 promoter, thus enhancing its transcription\u003csup\u003e\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e\u003c/sup\u003e. There are many TFs occupied on the promoter of FOSL1, including ELK-1, SRF, AP-1, and ATF/CREB\u003csup\u003e35\u003c/sup\u003e. Although we cannot formally exclude additional partners of mtp53, ELK-1 may be a significant recruitment factor because the consumption of ELK-1 impairs FRA-1 expression in BRAC.\u003c/p\u003e \u003cp\u003eIn light of the role of FRA-1 in metastasis, targeting FRA-1 in aggressive BRCA may be a new therapeutic option. This study offers a prism through which to look at the molecular basis of mtp53-driven metastasis and an identification scheme for a novel p53 mutational metastatic therapeutic target.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eData availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe datasets analyzed during the current study are available as follows. Gene expression microarray data can be accessed under GEO Accession No. GSE68248. ChIP-seq data were obtained from GEO Accession No. GSE66543 (MDA-MB-231) and ENCODE Project Consortium: A549 (ENCSR112XUO), HepG2 (ENCSR980EGJ), and Hela-s3 (ENCSR454DOC, ENCSR717QSS, ENCSR068MRQ).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eS.H. and H.L. designed the experiments, performed the statistical analysis, and wrote the manuscript. M.W., A. J., J.Y., R. G., X.L., and L.S performed experiments acquired. Z.Y. and Y.Z. assisted with writing the manuscript. All authors reviewed and approved the final version of the manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no competing interests.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eKandoth, C.\u003cem\u003eet al.\u003c/em\u003e Mutational landscape and significance across 12 major cancer types. \u003cem\u003eNature\u003c/em\u003e \u003cstrong\u003e502\u003c/strong\u003e, 333-339, doi:10.1038/nature12634 (2013).\u003c/li\u003e\n\u003cli\u003eRivlin, N., Brosh, R., Oren, M. \u0026amp; Rotter, V. Mutations in the p53 Tumor Suppressor Gene: Important Milestones at the Various Steps of Tumorigenesis. \u003cem\u003eGenes Cancer\u003c/em\u003e \u003cstrong\u003e2\u003c/strong\u003e, 466-474, doi:10.1177/1947601911408889 (2011).\u003c/li\u003e\n\u003cli\u003eTang, Q., Su, Z., Gu, W. \u0026amp; Rustgi, A. K. 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Bradykinin induces matrix metalloproteinase-9 expression and cell migration through a PKC-delta-dependent ERK/Elk-1 pathway in astrocytes. \u003cem\u003eGlia\u003c/em\u003e \u003cstrong\u003e56\u003c/strong\u003e, 619-632, doi:10.1002/glia.20637 (2008).\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"","lastPublishedDoi":"10.21203/rs.3.rs-1452900/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-1452900/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eTumor-related p53 mutations can provoke activities different from p53 wild-type tumors to lose tumor-suppressing function. Cells harboring p53 mutations possess a more aggressive property associated with highly pro-invasion, pro-metastasis, proliferation, and cell survival. By comparing the gene expression profiles of p53 mutant and p53 knockdown cancer cells, we figure out FRA-1 as a potential effector of mutp53-mediated metastasis. We demonstrate that the expression of FRA-1, a gatekeeper of mesenchymal-epithelial transition encoded by FOSL1, elevates in the presence of p53 mutations. Mechanistically, mutant p53 binds to ELK-1 in BRCA cells, implying their collaboration to induce FRA-1 expression. In short, this study deciphers new insights into how mtp53 handles metastasis.\u003c/p\u003e","manuscriptTitle":"Mutant p53 and ELK-1 co-drive the expression of FRA-1 in breast cancer cells","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2022-03-17 18:34:58","doi":"10.21203/rs.3.rs-1452900/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"f4b530a4-3589-486c-9f6e-c5563ae60957","owner":[],"postedDate":"March 17th, 2022","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2022-05-09T05:59:13+00:00","versionOfRecord":[],"versionCreatedAt":"2022-03-17 18:34:58","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-1452900","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-1452900","identity":"rs-1452900","version":["v1"]},"buildId":"-HB7Z8yhvgn0wM9Nzuekk","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}